Abstract
RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The ...adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.3 Å) and four holo cryo-electron microscopy structures (overall 3.0–3.5 Å, binding pocket 2.9–3.2 Å). The holo dimers exhibit global motions of helical twisting and bending around the dimer interface. A backbone comparison of the apo and holo states reveals a large structural difference in the P6 extension position. The central strand of the binding pocket, junction 6/3, changes from an ‘S’- to a ‘U’-shaped conformation to accommodate ligand. Furthermore, the binding pocket can partially form under 1 mM Mg2+ and fully form under 10 mM Mg2+ within the bound-like structure in the absence of ligand. Our results not only demonstrate the stabilizing ligand-induced conformational changes in and around the binding pocket but may also provide further insight into the role of the P6 extension in ligand binding and selectivity.
Graphical Abstract
Graphical Abstract
A single primary RNA sequence folds into heterogeneous conformations in both apo and holo states.
► We review the high resolution structures of the Notch receptor and ligands. ► Highlight the docking events of Notch receptor and ligand at the cell surface. ► Indicate the future challenges in ...understanding Notch receptor–ligand interactions.
The Notch receptor is part of a core signalling pathway which is highly conserved in all metazoan species. It is required for various cell fate decisions at multiple stages of development and in the adult organism, with dysregulation of the pathway associated with genetic and acquired diseases including cancer. Although cellular and in vivo studies have provided considerable insight into the downstream consequences of Notch signalling, relatively little is known about the molecular basis of the receptor/ligand interaction and initial stages of activation. Recent advances in structure determination of the extracellular regions of human Notch-1 and one of its ligands Jagged-1 have given new insights into docking events occurring at the cell surface which may facilitate the development of new highly specific therapies. We review the structural data available for receptor and ligands and identify the challenges ahead.
In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining ...strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C‐terminal domain (CTD) with homology to the metallo‐β‐lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+‐dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import.
During natural transformation in bacteria one DNA strand is transported into the cytoplasm via the membrane protein ComEC while the other strand is degraded. Here we show that the C‐terminal domain of ComEC has a nuclease activity that is important for natural transformation in B. subtilis. Thus, this domain is likely to be responsible for digesting the non‐transforming DNA strand.
Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal ...structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.
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► Crystal structure of C5b6 defines a platform for MAC assembly ► Cryo-EM map of sC5b9 reveals immune protection mechanism by host proteins ► MAC immune pores resemble those of bacterial toxins ► This assembly model provides a structural basis for fighting microbial infections
Activation of the mammalian complement system initiates the formation of membrane attack complexes that lyse microbes. Hadders, Bubeck, et al. reveal the stepwise buildup of the pore-forming complex and how host cells are protected from bystander damage. X-ray and cryo-EM data show how C6 captures C5b and how C7, C8αβγ, and C9 dock sideways onto C6, aligning their β-barrel forming domains in an arc. Vitronectin and clusterin enwrap the membrane perforating segments and block polymerization of C9.
Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a ...subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion.
The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the ...complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.
Cryo-EM diversifies Lea, Susan M.; Williams, Pamela A.
Current opinion in structural biology,
December 2022, 2022-Dec, 2022-12-00, 20221201, Letnik:
77
Journal Article
Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate ...complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the
virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex.
Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.
Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins ...(FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.
Tuberculosis (TB) is the leading cause of death from a single infectious agent and in 2019 an estimated 10 million people worldwide contracted the disease. Although treatments for TB exist, continual ...emergence of drug-resistant variants necessitates urgent development of novel antituberculars. An important new target is the lipid transporter MmpL3, which is required for construction of the unique cell envelope that shields Mycobacterium tuberculosis (Mtb) from the immune system. However, a structural understanding of the mutations in Mtb MmpL3 that confer resistance to the many preclinical leads is lacking, hampering efforts to circumvent resistance mechanisms. Here, we present the cryoelectron microscopy structure of Mtb MmpL3 and use it to comprehensively analyze the mutational landscape of drug resistance. Our data provide a rational explanation for resistance variants local to the central drug binding site, and also highlight a potential alternative route to resistance operating within the periplasmic domain.
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•Cryo-EM structure of M. tuberculosis MmpL3 determined at 3.0 Å resolution•An LMNG molecule within the periplasmic cavity suggests the TMM export pathway•Comprehensive structural mapping of resistance-conferring MmpL3 variants•Genome-mined MmpL3 mutations indicate minimal pre-existing resistance
Adams et al. report a structure of the M. tuberculosis glycolipid transporter MmpL3, a protein essential to envelope biogenesis and an emergent therapeutic target. The structure reveals the probable lipid export pathway, and mapping of resistance-conferring mutations suggests the existence of multiple routes to preclinical inhibitor evasion.
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